<p>Apoptosis is a conserved mode of cell death executed by a group of proteases named caspases, which collectively ensure tissue homeostasis in multicellular organisms by triggering a program of cellular "suicide" in response to developmental cues or cellular damage. </p><p>Accumulating evidence suggests that cellular metabolism impinges directly upon the decision to initiate cell death. Several links between apoptosis and metabolism have been biochemically characterized. Using <italic>Xenopus</italic> oocyte extracts, our laboratory previously discovered that caspase-2 is suppressed by NADPH metabolism through an inhibitory phosphorylation at S164. However, the physiological relevance of these findings has not been investigated at the whole organism level. Studies presented in this dissertation utilize both Schneider's <italic>Drosophila</italic> S2 (S2) cells and transgenic animals to untangle the influence of metabolic status on fly apoptosis.</p><p>We first demonstrate a novel link between <italic>Drosophila</italic> apoptosis and metabolism by showing that cellular NADPH levels modulate the fly initiator caspase Dronc through its phosphorylation at S130. Biochemically and genetically blocking NADPH production removed this inhibitory phosphorylation, resulting in the activation of Dronc and the subsequent apoptotic cascade in cultured S2 cells and specific neuronal cells in transgenic animals. Similarly, non-phosphorylatable Dronc was found to be more potent than wild-type in triggering neuronal apoptosis. Moreover, upregulation of NADPH prevented Dronc-mediated apoptosis upon abrogation of <italic>Drosophila</italic> Inhibitor of Apoptosis (IAP) protein 1 (DIAP1) by double-stranded RNA (dsRNA) or cycloheximide (CHX) treatment, revealing a novel mechanism of DIAP1-independent apoptotic regulation in <italic>Drosophila</italic>. Mechanistically, the CaMKII-mediated phosphorylation of Dronc hindered its activation, but not its catalytic activity. As NADPH levels have been implicated in the regulation of oocyte death, we demonstrate here that a conserved regulatory circuit also coordinates somatic apoptosis and NADPH levels in <italic>Drosophila</italic>.</p><p>Given the regulatory role of NADPH in the activation of Dronc in <italic>Drosophila</italic> and caspase-2 in vertebrates, we then attempted to further elucidate the underlying signaling pathways. By tracking the catabolic fate of NADPH, we revealed that fatty acid synthase (FASN) activity was required for the metabolic suppression of Dronc, as both the chemical inhibitor orlistat and FASN dsRNA abrogated NADPH-mediated protection against CHX-induced apoptosis in S2 cells. Interestingly, it has been previously demonstrated that blocking FASN induces cell death in numerous cancers, including ovarian cancer; however, the mechanism is still obscure. As our results predict that suppression of FASN activity may prevent the inhibitory phosphorylation of Dronc and caspase 2 (at S130 and S164 respectively), we examined the contribution of caspase-2 to cell death induced by orlistat using ovarian cancer cells. Indeed, caspase-2 S164 was dephosphorylated upon orlistat treatment, initiating the cleavage and activation of caspase-2 and its downstream target, Bid. Knockdown of caspase-2 significantly alleviated orlistat-induced cell death, further illustrating its involvement.</p><p>Lastly, we developed an assay based on bimolecular fluorescence complementation (BiFC) to monitor the oligomerization of Dronc in S2 cells, a crucial step in its activation. The sensitivity of this assay has been validated with several apoptotic stimuli. A future whole-genome screen employing this assay is planned to provide new insights into this complex apoptotic regulatory network by unbiasedly identifying novel apoptotic regulators.</p> / Dissertation

Cocaethylene (CE) is a cocaine metabolite formed during alcohol and cocaine co-consumption. To our knowledge, no previous studies were conducted assessing CE as a biomarker indicating chronic excessive alcohol consumption in a suspected high-risk population. In this study, we hypothesized that hair CE can be an effective marker for alcohol consumption in a high-risk population. We recorded cocaine, benzoylecgonine, and CE levels in hair samples from individuals, establishing the predictive value of CE by comparing it to hair levels of the widely used hair fatty acid ethyl esters (FAEE), direct markers of chronic excessive alcohol consumption. CE had 14.04% sensitivity and 95.18% specificity in samples separating FAEE positive/negative results. The positive predictive value was 0.66, showing that the results for individuals with CE positive results were more than likely to be FAEE positive, but not conclusively. Thus, CE cannot be used as a definitive marker, indicating chronic excessive alcohol consumption.

Canola is a cool-season, oilseed crop grown throughout Europe, Canada, and the Northern Great Plains region of the United States. The expansion of canola production into new growing regions, such as the Southern Plains region, has resulted in new production challenges. The Southern Plains region cultivates canola as a winter annual compared to a spring annual for the Northern Great Plains and Canada. Given the difference in climate and weed spectrum, region-specific weed management systems need to be developed. Agronomic practices can affect seed oil content, protein content, and fatty acid composition, however the effect of herbicides on these and other characteristic of canola are unknown. Therefore, experiments were conducted in 2010 and 2011 to evaluate a broad spectrum of herbicides for potential use in South Texas canola production with respect to crop injury, effects on canola seed oil content, fatty acid composition, weed control, biomass yield, and forage quality. Visual crop injury at 42 DAE was unacceptable for saflufenacil at both 0.12 and 0.06 kg ai ha-1 and ethalfluralin at 1.05 kg ai ha-1. Trifluralin at 1.12 and 0.56 kg ai ha-1, S-metolachlor at 2.14 and 1.07 kg ai ha-1, pyroxasulfone at 0.24 and 0.12 kg ai ha-1, and pendimethalin at 0.8 kg ai ha-1 had lowest visual injury of all treatments. Fluroxypyr applied EPOST caused severe injury at both 0.21 and 0.11 kg ae ha-1. All other EPOST treatments did not cause any visible injury. Seed oil content was not affected by the herbicides evaluated. Fatty acid composition, specifically stearic acid, oleic acid, linolenic acid, and oleic to linolenic acid ratio, was affected by herbicide treatments. This research found that protoporphyrinogen oxidase (PPG oxidase) inhibitor herbicides, such as carfentrazone-ethyl and saflufenacil, negatively affect canola oil quality. Biomass yield was improved for all herbicide treatments except pendimethalin PRE when compared to the untreated plots. Crude protein content of canola forage was not affected by herbicide treatment. Digestible dry matter appeared to be reduced by treatments that included an EPOST application of sethoxydim. The research shows that pendimethalin and S-metolachlor may be suitable for canola production in South Texas based on low crop injury and effective weed control. Neither pendimethalin nor S-metolachlor is currently labeled for use in canola. The herbicides trifluralin, ethalfluralin, quizalofop P-ethyl, ethametsulfuron-methyl, sethoxydim, glyphosate, clethodim, and clopyralid are currently labeled for use in canola and were confirmed suitable for canola production in South Texas. Carfentrazone-ethyl is currently labeled for use in canola but the effects on oil quality should be considered.

This thesis presents the cloning, purification, crystallization, and structural studies of two unknown proteins from Mycobacterium tuberculosis, and of an aminotransferase from Mycobacterium smegmatis. Structural knowledge of these proteins is of highest interest for structure-based drug design, which is one of the approaches that can be used in order to fight tuberculosis (TB). The structure of the conserved hypothetical protein Rv0216 was refined to a resolution of 1.9 Å. The structure exhibits a so-called double hotdog-fold, similar to known hydratases. However, only parts of the hydratase active site are conserved in Rv0216, and no function could be assigned to the protein. Several Rv0216-like protein sequences were found in a variety of actino- and proteobacteria, suggesting that these proteins form a new protein family. Furthermore, other hotdog-folded proteins in M. tuberculosis were identified, of which a few are likely to be hydratases or dehydratases involved in the fatty acid metabolism. The structure of Rv0130 exhibits a single hotdog-fold and contains a highly conserved R-hydratase motif. Rv0130 was shown to hydrate fatty acid coenzyme A derivatives with a length of six to eight carbons. The Rv0130 active site is situated in a long tunnel, formed by a kink in the central hotdog-helix, which indicate that it can utilize long fatty acid chains as well. A number of previously predicted hotdog-folded proteins also feature a similar tunnel. The structure of branched chain aminotransferase (BCAT) of M. smegmatis was determined in the apo-form and in complex with an aminooxy inhibitor. Mycobacterial BCAT is very similar to the human BCAT, apart for one important difference in the active site. Gly243 is a threonine in the human BCAT, a difference that offers specificity in inhibition and substrate recognition of these proteins. The aminooxy compound and MES were found to inhibit the mycobacterial BCAT activities. The aminooxy compound inhibits by blocking the substrate-pocket. A second inhibitor-binding site was identified through the binding of a MES molecule. Therefore, both the MES-binding site and the substrate-pocket of M. smegmatis BCAT are suggested to be potential sites for the development of new inhibitors against tuberculosis.

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells.

The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375755 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Global studies show the effect of polymorphism of selected genes on dairy production and fatty acid spectrum. The aim of the diploma thesis was genotyping of candidate FASN locus with a focus on milk yield and fatty acid spectrum depending on genotype. The thesis describes the characteristics of cow's milk, milk fat and fatty acids in milk fat. Furthermore, the thesis deals with the genome of cattle and the potential influence of polymorphism of candidate genes affecting fatty acids of milk fat. Genotypes for FASN were determined by the PCR-RFLP method, the milk yield of selected dairy cattle was statistically evaluated according to the milk production indices for 1st lactation in individual breeds, and the determination of fatty acids was performed by spectrophotometry followed by statistical evaluation. The resulting genotypes in selected breeds were only two, the GG genotype with a higher relative frequency than the genotype AG. There is no statistically significant difference between FASN genotypes depending on the milk yield and the fatty acids spectrum.

L'obésité et les maladies du foie associées (NAFLD) augmentent le risque et la sévérité de l’hépatotoxicité induite par certains xénobiotiques, mais les mécanismes impliqués sont encore mal compris. Pour l'éthanol et le paracétamol (APAP), le rôle du cytochrome P450 2E1 (CYP2E1) hépatique est suspecté car l'activité de cette enzyme est augmentée au cours de ces pathologies dysmétaboliques. Le 1er objectif de notre travail expérimental a été de mettre au point un modèle cellulaire de NAFLD caractérisé non seulement par l'accumulation de triglycérides mais aussi par l’augmentation de l'activité du CYP2E1. Pour cela, des cellules humaines HepaRG différenciées ont été incubées pendant une semaine avec de l'acide stéarique ou de l'acide oléique, en présence de 3 concentrations différentes d'insuline. Les triglycérides cellulaires et l'expression de gènes induits au cours de la stéatose étaient similaires avec les deux acides gras. Cependant, l'activité du CYP2E1 était significativement augmentée uniquement par le stéarate et ceci était associé à une diminution de l'activité du CYP3A4, une autre caractéristique des NAFLD. L’activité du CYP2E1 dans les cellules HepaRG était réduite par l'insuline d'une manière concentration-dépendante et cet effet était reproduit sur des hépatocytes humains en culture primaire. Ainsi, l'activité du CYP2E1 était la plus élevée dans les cellules HepaRG cultivées avec du stéarate et sans insuline. Le 2ème but de notre étude était ensuite d'évaluer la cytotoxicité de l’APAP sur des cellules HepaRG présentant ou non une stéatose et une induction du CYP2E1. Des expériences avec une large gamme de concentrations d’APAP (de 1 à 20 mM) indiquaient que la perte cellulaire d'ATP et du glutathion (GSH) était presque toujours plus forte en présence de stéarate. Dans les cellules prétraitées avec le chlorméthiazole (CMZ, un inhibiteur du CYP2E1), la moindre diminution d’ATP était plus importante en présence de stéarate, avec de faibles (2,5 mM) ou de fortes (20 mM) concentrations d’APAP. Cependant, en l'absence d'insuline, la moindre chute d’ATP induite par le CMZ était significativement plus forte uniquement pour 20 mM d’APAP. Étonnamment, suite au prétraitement par le CMZ, il n'y avait pas de protection vis-à-vis de la diminution du GSH et de la formation des adduits APAP-protéines. Enfin, les concentrations du métabolite APAP-glucuronide étaient significativement augmentées en présence d'insuline. Ainsi, lorsqu’elle est étudiée dans des conditions spécifiques de culture, la lignée cellulaire HepaRG semble être un modèle intéressant de NAFLD, notamment en ce qui concerne les activités du CYP2E1 et du CYP3A4. Nos données suggèrent aussi que l’induction du CYP2E1 observée au cours des NAFLD pourrait être secondaire à l'accumulation de certains acides gras et à la présence d’une faible signalisation insulinique dans le foie. Ainsi, ce modèle cellulaire peut être utilisé pour mettre en évidence les principaux facteurs métaboliques et hormonaux favorisant hépatotoxicité de l’APAP chez les personnes obèses. Cette thèse inclut également une revue de la littérature sur l’hépatotoxicité de l’APAP dans le contexte de l’obésité et des NAFLD (Michaut et al., Liver Int 2014). / Obesity and nonalcoholic fatty liver disease (NAFLD) are able to increase the risk and the severity of hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are still poorly understood. For toxic compounds such as ethanol and acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during obesity and NAFLD. The first aim of our experimental study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, differentiated human HepaRG cells were incubated during one week with stearic acid, or oleic acid, in the presence of 3 different concentrations of insulin. Cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids. However, CYP2E1 activity was significantly increased only by stearate and this was associated with lower CYP3A4 activity, another metabolic feature reported in NAFLD. CYP2E1 activity in HepaRG cells was reduced by insulin in a concentration-dependent manner and this effect was reproduced in cultured primary human hepatocytes. Hence, the highest CYP2E1 activity was observed in HepaRG cells with stearate and without insulin. Next, the second aim of our study was to assess APAP cytotoxicity in HepaRG cells presenting or not lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations (1 to 20 mM) showed that the cellular loss of ATP and glutathione (GSH) was almost always stronger in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole (CMZ), recovery of cellular ATP was significantly higher in the presence of stearic acid with both low (2.5 mM) and high (20 mM) concentrations of APAP. However, in the absence of insulin, CMZ-induced ATP recovery was significantly greater only for 20 mM of APAP. Surprisingly, there was no recovery of cellular GSH and no reduction of APAP-protein adducts following CMZ pretreatment. Finally, levels of APAP-glucuronide were significantly enhanced in the presence of insulin. Hence, when studied in specific conditions of culture, the HepaRG cell line can be a valuable model of human NAFLD, especially regarding CYP2E1 and CYP3A4 activity. Our data also suggest that higher CYP2E1 activity in NAFLD could be secondary to the hepatic accumulation of some fatty acids and to the presence of low insulin signaling. This cellular model can be thus used to unveil the main metabolic and hormonal factors favoring APAP hepatotoxicity in obese individuals. This thesis also includes a review on APAP hepatotoxicity in the context of obesity and NAFLD (Michaut et al., Liver Int 2014).

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Methylmalonic acidemias are inherited metabolic disorders characterized by methylmalonate (MMA) accumulation and neurological dysfunction, including seizures. Dietary fatty acids are known as an important energy source and reduce seizure activity in selected acute animal models. This study investigates whether the chronic treatment with fish oil or with oleic acid attenuates MMA-induced seizures. Adult male Wistar rats were treated with fish oil (85 mg/kg), oleic acid (85 mg/kg) or vehicle (0.42 % aqueous Cremophor EL , 4 mL/kg/body weight/day), p.o., for 75 days. In the 73th day were implanted a cannula in the right lateral ventricle with electrodes over the parietal cortex for EEG recording. In the 76th day half the animals from each group were injected with NaCl (2.5 μmol/2.5 μL, i.c.v.), and the other half with MMA (2.5 μmol/2.5 μL, i.c.v.), and seizure activity was measured by EEG recording with concomitant behavior monitoring. The effect of prostaglandin E2 (PGE2) on Na+,K+-ATPase activity of slices of cerebral cortex from NaCl-injected (control) animals was determined. Fish oil administration increased the latency for MMA-induced tonic-clonic seizures and reduced the mean amplitude of ictal EEG recordings. Oleic acid decreased mean amplitude of ictal EEG recordings. Treatment with fish oil prevented PGE2-induced decrease of Na+,K+-ATPase activity in cortical slices in vitro. The results support a major anticonvulsant role for fish oil against MMA-induced seizures. The decreased sensitivity of Na+,K+-ATPase from fish oil-treated animals to the inhibitory effect of PGE2 may be related to its currently reported anticonvulsant activity. / A acidemia metilmalônica é um erro inato do metabolismo caracterizado pelo acúmulo tecidual de ácido metilmalônico (MMA) e disfunção neurológica, incluindo convulsões. Os ácidos graxos dietéticos são conhecidos como fonte de energia e reduzem a atividade convulsivante em determinados modelos experimentais agudos de convulsão. Este estudo investiga se o tratamento crônico com óleo de peixe ou com ácido oléico atenua as convulsões induzidas por MMA. Ratos Wistar machos adultos foram tratados com óleo de peixe (85 mg / kg), ácido oléico (85 mg / kg) ou veículo (solução aquosa de Cremophor EL® 0,42%, 4 mL / kg de peso corporal / dia), via oral, por 75 dias. No 73º dia foi implantada uma cânula no ventrículo lateral direito e dois eletrodos sobre o córtex parietal para o registro eletroencefalográfico. No 76º dia metade dos animais de cada grupo foi injetada com NaCl (2,5 Smol / 2,5 SL, i.c.v.), e outra metade com MMA (2,5 Smol / 2,5 SL, i.c.v.), e a atividade convulsiva foi medida por EEG e monitoramento comportamental concomitantemente. O efeito da prostaglandina E2 (PGE2) na atividade Na+, K+-ATPase foi determinada em fatias de córtex cerebral dos animais injetados com NaCl (controle). A administração de óleo de peixe aumentou a latência para as convulsões tônico-clônicas induzidas por MMA e reduziu a amplitude média dos registros ictais de EEG. O ácido oléico diminuiu a amplitude média dos registros ictais de EEG. O tratamento com óleo de peixe preveniu a diminuição da atividade da Na+, K+-ATPase induzida por PGE2 em fatias corticais in vitro. Os resultados suportam um papel importante anticonvulsivante do óleo de peixe sobre as convulsões induzidas por MMA. A prevenção da redução da Na+, K+-ATPase induzida por PGE2 nos animais tratados com óleo de peixe pode estar relacionada à sua atividade anticonvulsivante atualmente relatada.

Três experimentos foram conduzidos com o objetivo de avaliar os efeitos do fornecimento de baixos teores de óleo de peixe em substituição parcial ao óleo de soja, sobre o consumo de matéria seca (CMS), produção e perfil de ácidos graxos do leite de ovelhas, ganho médio diário de peso corporal (GMD), características da carcaça e composição de ácidos graxos da carne de cordeiros, digestibilidade dos nutrientes, características de fermentação ruminal e metabolismo ruminal dos ácidos graxos. Os tratamentos consistiram de uma dieta controle (CONT), sem adição de óleo; e 4 dietas adicionadas com 4,0% de óleo, consistindo de 0,0% (0P); 0,25% (25P); 0,50% (50P) e 0,75% de óleo de peixe (75P), com o óleo de soja completando o teor de 4,0% de óleo adicionado (% MS). No Experimento I a dieta controle foi composta por 70% de concentrado e 30% de volumoso, nos Experimentos II e III a dieta controle foi composta por 90% de concentrado e 10% de volumoso. Experimento I: Foram utilizadas 50 ovelhas, distribuídas em delineamento experimental de blocos completos casualizados. Verificouse aumento linear na produção de leite das ovelhas e no GMD das crias com a inclusão de óleo de peixe nas dietas. As concentrações de ácido vacênico, CLA C18:2 trans-10, cis-12, ácido eicosapentaenóico (EPA) e ácido docosahexaenóico (DHA) também aumentaram linearmente com os teores crescentes de inclusão de óleo de peixe. Experimento II: Foram utilizados 50 cordeiros, distribuídos em delineamento experimental de blocos completos casualizados. O CMS expresso em % do peso corporal (PC) e em g/kg de PC0,75 aumentou linearmente com os teores crescentes de inclusão de óleo de peixe, o que resultou em aumento linear no GMD dos cordeiros. A concentração de ácido esteárico reduziu com os teores crescentes de substituição do óleo de soja pelo óleo de peixe. Verificou-se aumento linear na concentração de ácido vacênico à medida que o óleo de peixe foi adicionado à dieta. Em comparação ao tratamento controle, os animais alimentados com as dietas contendo óleo de peixe apresentaram maior concentração de CLA C18:2 cis-9, trans-11 na carne. Experimento III: Foram utilizados cinco borregos, canulados no rúmen e no duodeno, distribuídos em delineamento experimental quadrado latino 5 x 5. A suplementação com as fontes de óleo reduziu a digestibilidade da PB. A concentração de acetato, butirato e dos ácidos graxos de cadeia curta (AGCC) totais foi maior no conteúdo ruminal dos animais alimentados com a dieta controle em relação aos das dietas contendo óleo, como conseqüência, o pH ruminal destes animais foi inferior. O fluxo duodenal de C18:1 trans-11 e CLA C18:2 cis-9, trans-11 foi superior para os animais que receberam gordura suplementar. Observou-se aumento linear no fluxo duodenal de ácido C18:1 trans-11 em resposta a inclusão de óleo de peixe nas dietas. A inclusão de 0,75% de óleo de peixe na dieta misturado à 3,25% de óleo de soja mostrou-se como a melhor alternativa avaliada. / Three trials were conducted to evaluate the effects of small amounts of fish oil supply in partial replacement of soybean oil on dry matter intake (DMI), lactation performance and milk fatty acid composition of ewes, growth, carcass characteristics, and on meat fatty acid composition of feedlot lambs, some rumen constituents, and ruminal fatty acid metabolism. Treatments consisted of a control diet (CONT), and 4 diets with 4% added fat consisting of 0.0% (0FO), 0.25% (25FO), 0.50% (50FO) and 0.75% (75FO) fish oil with soybean oil providing the balance of 4% added fat. In trial I the control treatment consisted of 30:70 ratio of forage to concentrate (DM basis). In trials II and III the control treatment consisted of 10:90 ratio of forage to concentrate (DM basis). Trial I: Fifty Santa Inês ewes were penned individually and used in a randomized complete block design. Milk production and preweaning ADG of lambs increased linearly when fish oil replaced soybean oil. Vaccenic acid, CLA trans-10, cis-12, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased linearly with fish oil inclusion. Trial II: Fifty Santa Inês ram lambs were penned individually and used in a randomized complete block design. DMI (% of BW and g/kg of BW0,75) increased linearly when fish oil replaced soybean oil, as consequence ADG also increased. Stearic acid concentration decreased and vaccenic acid increased with fish oil inclusion. CLA C18:2 cis-9, trans-11 showed higher concentration in meat of animals fed diets containing fish oil compared to the control diet. Trial III: Five ram lambs cannulated in the rumen and proximal duodenum were assigned in a 5 x 5 Latin Square design. Soybean oil and fish oil supplementations decreased CP digestibility. Ruminal concentrations of acetate, butyrate and total SCFA were higher for animals fed the control diet. Ruminal pH was lower for animals fed the control diet compared to diets with oils. Duodenal flow of C18:1 trans-11 and CLA C18:2 cis-9, trans-11 was greater for diets containing supplemented oils. C18:1 trans-11 flow to the duodenum increased linearly with fish oil inclusion.The inclusion of 0.75% of fish oil in the diet mixed with 3.25% soybean oil was the best alternative evaluated.